KR20230044133A - Cancer vaccine composition comprising peptides derived from tumor-associated antigen, and adjuvant consisting of lipopeptide and an immunoactive substance - Google Patents

Abstract

The present invention relates to an anti-cancer vaccine composition comprising [tumor-associated antigen (TAA)-derived peptide] and [adjuvant composed of lipopeptide and immune-activating substance]. Specifically, the tumor-related antigen-derived peptide of the present invention is a peptide that specifically binds to human leucocyte antigen (HLA), and a vaccine composition is manufactured by mixing the peptide combination with the above characteristics with the adjuvant at an optimal ratio, and the vaccine composition is used for the prevention or treatment of cancer.

Description

Cancer vaccine composition comprising peptides derived from tumor-associated antigen, and adjuvant consisting of lipopeptide and an immunoactive substance}

The present invention relates to an anti-cancer vaccine composition comprising an adjuvant composed of peptides and lipopeptides derived from tumor-associated antigens and immunoactive substances. Specifically, the present invention provides a peptide that specifically binds to human leucocyte antigen (HLA), a vaccine for cancer treatment comprising the peptide, a lipopeptide, and a poly(I:C) adjuvant, and a vaccine for the treatment of cancer It relates to the use of a pharmaceutical composition containing a vaccine as an active ingredient as a preventive or therapeutic agent for cancer.

Compared to normal cells, cancer is characterized by unregulated cell growth, invasion into nearby tissues, and sometimes metastasis to distant tissues. In modern times, with the development of scientific medical technology, many diseases have become treatable and incurable diseases have almost disappeared. However, unlike other diseases, cancer requires very complex and difficult treatment, and even that treatment is not completely effective. .

Anticancer treatment physically removes cancer tissue, or through irradiation or administration of anticancer drugs, anti-apoptotic molecules, telomerase, growth factor receptor gene, and signaling It is performed by inhibiting the expression of genes necessary for the survival of cancer cells, such as signaling molecules, or by inducing apoptosis (Knudson AG, Nature reviews. Cancer 1(2): 157-162, 2001).

However, in this treatment process, not only cancer cells but also normal cells can be affected, causing side effects. Accordingly, the need for an anti-cancer vaccine as an alternative method of treating cancer is emerging.

Anticancer vaccines are vaccines that educate the human immune system to remember cancer cell-specific antigens to attack cancer cells, and there are various types of antigens and antigen delivery methods (eg, peptide, DNA, RNA, dendritic cells). Vaccines for cancer treatment should induce an immune response against self-antigens rather than non-self-antigens, unlike traditional prophylactic vaccines that prevent viral or bacterial infection from outside. In addition, the vaccine should work when administered after cancer is formed and should exert an immune effect to suppress cancer, and since the expression rate of each cancer antigen is different even in patients with the same carcinoma, as many types of cancer antigens as possible can be used. must be presented together.

In fact, many types of tumor-associated antigens are immune tolerogens as self-antigens, making it difficult to induce a specific immune response. These limitations can be overcome by inducing an immune response against antigens commonly expressed in many cancer types or antigens expressed in a single cancer type. Recently, antigens that can act in common in various cancer types and antigens that are highly expressed in one cancer type have been reported.

The tumor-associated antigen (TAA), which is a target in anti-cancer vaccines, is classified as follows. A) Cancer-testis antigen: Antigens belonging to this group are not expressed in normal tissues except for testicular spermatocytes/spermatocytes, but are highly expressed in cancer tissues, so they are initially cancer-testis antigens. It was called the testicular (CT) antigen. Because testicular cells do not express class I or II HLA molecules, these antigens cannot be recognized by T cells on normal cells and can therefore be considered immunologically tumor-specific. Well-known examples of CT antigens are MAGE members and NY-ESO-1. B) Differentiation antigen: TAA is an antigen in which a protein expressed in differentiating cells of a specific tissue is shared between a tumor and a normal cell in which a tumor has occurred. A representative example of a differentiation antigen is Melan-A/MART-1 found in melanoma and normal melanocytes. This melanocyte lineage-associated protein is involved in the biosynthesis of melanin and is not tumor specific, but is widely used in cancer immunotherapy. Another example is tyrosinase for melanoma or prostate-specific antigen (PSA) for prostate cancer. C) Overexpressed TAA: A TAA that generally shows low expression levels in normal tissues and is widely expressed in other types of tumors. Many of the peptide epitopes processed and potentially presented by antigen presenting cells (APCs) in normal tissues may be below the threshold level for T cell recognition and therefore do not induce an immune response in normal cells. However, overexpression in tumor cells can disrupt previously established resistance and initiate an anticancer response. Prominent examples of TAAs in this group are Her-2/neu, Survivin, Telomerase, or WT-1 (Wilms' tumor 1). D) Tumor-specific antigen: These unique TAAs arise as mutations in normal genes (β-catenin, CDK4, EGFRvIII, etc.). Some of these molecular alterations have been associated with tumor transformation and/or progression. Tumor-specific antigens are generally capable of eliciting a strong immune response without the risk of an autoimmune response to normal tissue. On the other hand, in most cases tumor-specific antigens are expressed only in specific cancers, not generally shared among many individual cancers. E) TAAs arising from abnormal post-translational modifications: These TAAs may occur in proteins that are neither specific nor overexpressed, but are associated with tumors by post-translational processes that are predominantly active in tumors. Examples of this class are events such as protein splicing during degradation processes, which may or may not be tumor specific, or in the case of MUC-1 (Mucin 1), altered glycosylation patterns leading to new epitopes in the tumor. Occurs.

There is a method of using tumor cell lysate, protein, DNA/RNA, plasmid, and T cell epitope peptide as an antigen form of an anti-cancer vaccine. Among them, the peptide epitope is the most effective substance for killing cancer cells or inhibiting the growth of cancer cells by activating antigen-specific T cells. In addition, there are advantages in that the safety of the peptide itself is high, the price is low, the production is easy, and a combination of various antigens is possible.

Anticancer vaccines target peptide epitopes derived from tumor-associated antigens, which are presented by molecules of the major histocompatibility complex (MHC). In humans, the MHC molecule is specified as human leucocyte antigen (HLA). There are two classes of MHC-molecules, MHC class I and MHC class II. MHC class I molecules are composed of α chain and beta- ₂ -microglobulin chain, and MHC class II molecules are composed of α chain and β chain. Their three-dimensional shape forms a binding groove, which is used for non-covalent binding with peptides.

MHC class I molecules are present in most nucleated cells and represent endogenous proteins, defective ribosomal products (DRIPs) and peptides produced by proteolytic cleavage of large peptides. However, peptides derived from endosome compartments or from exogenous sources are also found in MHC class I molecules. Class I presentation in this non-classical manner is referred to in the literature as cross-presentation (Brossart and Bevan, Blood , 1997).

MHC class II molecules are mostly found on specialized antigen-presenting cells (APCs), which present peptides of foreign or transmembrane proteins that are mainly taken up by antigen-presenting cells. Complexes of peptides and MHC class I molecules are recognized by CD8-positive (CD8+) T cells with the appropriate T-Cell Receptor (TCR), whereas complexes of peptides and MHC class II molecules do not bind to the appropriate TCR. It is recognized by CD4-positive helper T cells (CD4+ helper T cells). CD4-positive helper T cells play an important role in effectively inducing and sustaining the response of CD8-positive cytotoxic T cells. Therefore, CD4-positive T-cell epitopes derived from tumor-associated antigens are very important for the development of drugs that induce anti-tumor immune responses (Gnjatic et al., PNAS , 2003).

Helper T cells support the surrounding environment through cytotoxic T lymphocyte (CTL)-friendly cytokines at the tumor site and effector cells such as CTL, natural killer cells, macrophages, Induce granulocytes (Tay et al., Cancer Gene Therapy , 2020). Helper T cells activated by MHC class II play an important role in regulating CTL effector function in anti-tumor immunity. Helper T cell epitopes that trigger helper T cell responses of TH1-type supporting effector functions of CD8-positive killer T cells contain cytotoxic functions against tumor cells that display tumor-associated peptide/MHC complexes on their cell surface. In this way, the tumor-associated helper T cell peptide epitope can be used as an active pharmaceutical ingredient in a vaccine composition to stimulate an anti-tumor immune response, either alone or in combination with other tumor-associated peptides. For example, in mammalian animal models such as mice, even in the absence of CD8-positive T lymphocytes, CD4-positive T cells suppress tumors through inhibition of angiogenesis by secretion of interferon gamma (IFN-g). It is known to inhibit (Beatty and Paterson, Immunologic Research , 2001; Mumberg et al., PNAS , 1999).

CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or CD4-positive helper T cells (ligand: MHC class II molecule + peptide epitope) is important for the development of tumor vaccines. Therefore, in the present invention, a peptide epitope recognizing MHC class I molecules and a peptide epitope recognizing MHC class II molecules were used together.

Basically, any peptide capable of binding to an MHC molecule can function as a T cell epitope. Prerequisites for eliciting a T cell response in vitro or in vivo are the presence of T cells with the corresponding TCR and the absence of immune tolerance against this specific epitope.

Identification of genes that are overexpressed in tumor tissues or human tumor cell lines or that are selectively expressed in such tissues or cell lines do not provide precise information about the use of antigens transcribed from these genes in immunotherapy. This is because only individual subpopulations of epitopes of these antigens are suitable for such applications, since T cells with corresponding TCRs must appear and there should be no or minimal immune tolerance to this particular epitope. Therefore, it is important to select only overexpressed or selectively expressed peptides that appear to be associated with MHC molecules while minimizing immune tolerance of T cells and maximizing function and proliferation.

However, since the number of MHC alleles is too large, it is practically inefficient to select peptides that bind strongly and stably by directly testing candidate peptides for all alleles. Instead, peptides capable of strongly binding to the three-dimensional binding grooves present in various MHC molecules can be effectively predicted using an in silico algorithm program or database.

These databases use information on peptide motifs related to the binding of MHC and peptides and databases obtained from mass spectrometers after eluting peptides bound to each MHC molecule to analyze various peptide epitopes in a specific protein sequence. can be predicted and found.

Representative databases include SYFPEITHI, MHCPEP, NetMHCpan-4.1EL, and NetMHCIIpan-4.0EL. However, all peptides predicted by avidity do not effectively induce T cell responses in vitro or in vivo . Although the binding ability predicted by the computer program was high, it may not induce sufficient immunogenicity in vitro or in vivo, and rather may induce immune tolerance.

Therefore, in order for these predicted peptides to be used as anticancer vaccines, it is necessary to select the most effective and safe peptides through practical experiments to determine whether they cause effective immunogenicity in vitro or in vivo and whether they induce immune tolerance. Experiments require a lot of effort in terms of money, material, and time. Therefore, in the present invention, peptides that are related to MHC class I and II molecules in various cancer antigens, are known to be able to maximize T cell function and proliferation, and have already been secured in safety in various previous clinical trials were preferentially selected. .

In order to increase the activation and function of T cells against tumor-associated antigens, the binding of TCR and peptides is primarily important, but the results of previous clinical trials using actual peptides as vaccines did not show the expected effect. The biggest reason is that the peptide alone failed to induce sufficient immunogenicity. For effective immunogenicity, costimulatory signals by antigen-presenting cells and the help of appropriate cytokines are required, otherwise T cells may be anergic and immune tolerance may be induced. Therefore, an adjuvant is essential to induce appropriate and sufficient T cell activation and elicit an anticancer immune response therethrough.

The present invention uses an adjuvant composed of a lipopeptide and an immunoactive substance as a composition of an anticancer vaccine. This adjuvant induces humoral and cellular immune responses excellently, and particularly increases the Th1 type immune response, which is important for anticancer responses. Therefore, the present inventors confirmed that an adjuvant composed of a lipopeptide and an immunoactive substance helps cancer antigen-specific T cell activation by the peptide and more effectively induces a tumor-associated antigen-specific immune response. completed the present invention.

An object of the present invention is to provide an anti-cancer vaccine composition comprising [peptides derived from tumor-associated antigens].

Another object of the present invention is to provide an anti-cancer vaccine composition comprising [a peptide derived from a tumor-associated antigen] and [an adjuvant composed of a lipopeptide and an immunoactive substance].

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising the anti-cancer vaccine composition as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, which is used in combination with one or more treatments selected from the group consisting of the anti-cancer vaccine composition and chemo-cancer agents, targeted anti-cancer agents, immune checkpoint inhibitors and radiation therapy will be.

In order to achieve the above purpose,

The present invention provides an anti-cancer vaccine composition comprising [a peptide derived from one or more tumor-associated antigens selected from the group consisting of hTERT, MAGE-A3, MUC-1, Survivin, and WT-1].

In addition, the present invention is [peptide derived from one or more tumor-associated antigens selected from the group consisting of hTERT, MAGE-A3, MUC-1, Survivin and WT-1] and [adjuvant consisting of lipopeptide and immunoactive substance It provides an anti-cancer vaccine composition comprising ].

The [peptides derived from tumor-associated antigens] include any one or more of the following peptides.

i) the peptide of SEQ ID NO: 1 derived from hTERT and of type HLA-A02 (MHC Class I) (SEQ ID NO: 1: ILAKFLHWL);

ii) the peptide of SEQ ID NO: 2 derived from hTERT and of type HLA-A24 (MHC Class I) (SEQ ID NO: 2: VYAETKHFL);

iii) a peptide of SEQ ID NO: 3 derived from hTERT and of type HLA-DRB1 (MHC Class II) (SEQ ID NO: 3: EARPALLTSRLRFIPK);

iv) the peptide of SEQ ID NO: 4 derived from hTERT and of type HLA-DRB1 (MHC Class II) (SEQ ID NO: 4: RPGLLGASVLGLDDI);

v) a peptide of SEQ ID NO: 5 derived from Survivin and of type HLA-A02 (MHC Class I) (SEQ ID NO: 5: ELTLGEFLKL);

vi) a peptide of SEQ ID NO: 8 derived from Mage-A3 and of type HLA-A02 (MHC Class I) (SEQ ID NO: 8: KVAELVHFL);

vii) a peptide of SEQ ID NO:9 derived from Mage-A3 and of type HLA-A24 (MHC Class I) (SEQ ID NO:9: IMPKAGLLI);

viii) the peptide of SEQ ID NO: 12 derived from MUC-1 and of type HLA-A02 (MHC Class I) (SEQ ID NO: 12: TAPPVHNV);

ix) a peptide of SEQ ID NO: 15 (SEQ ID NO: 15: RMFPNAPYL) derived from WT-1 and of type HLA-A02 (MHC Class I); and

x) Peptide of SEQ ID NO: 16 derived from WT-1 and of type HLA-DRB1 (MHC Class II) (SEQ ID NO: 16: KRYFKLSHLQMHSRKH).

The adjuvant is prepared by mixing a lipopeptide and an immunoactive substance.

The adjuvant includes liposomes having lipopeptides inserted on the surface and immunoactive substances.

The liposomes are composed of lipids.

The immunoactive substance is at least one selected from the group consisting of poly(I:C), QS21, MPLA, CpG, and flagellin.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the anti-cancer vaccine composition as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, wherein the anti-cancer vaccine composition is used in combination with one or more treatments selected from the group consisting of chemo-cancer agents, targeted anti-cancer agents, immune checkpoint inhibitors, and radiation therapy.

The cancer tumor-associated antigen-derived peptide provided in one aspect of the present invention can effectively induce anti-cancer immunity, and an anti-cancer vaccine composition comprising a tumor-associated antigen-derived peptide, a lipopeptide, and an adjuvant composed of an immunoactive substance can be used in a variety of ways. It can effectively induce anti-cancer immune action against carcinoma, and can be usefully used for preventing and treating cancer by significantly enhancing the anti-cancer effect in combination with existing anti-cancer treatments such as chemo-cancer drugs with different mechanisms, immune checkpoint inhibitors, and radiation therapy. there is.

1 is a result of confirming the immunogenicity of the tumor-associated antigen-derived single peptide provided in the present invention using ELISPOT analysis.
Figure 2 is the result of confirming the immunogenicity using ELISPOT analysis when the tumor-associated antigen-derived peptides provided in the present invention are combined for each antigen.
Figure 3 is the result of confirming immunogenicity using ELISPOT analysis when the tumor-associated antigen-derived peptides provided in the present invention are combined according to the number of peptides.
Figure 4a shows the CHA-2 (IL-2 20U / ml) peptide alone (5ug / ml or 50ug / ml), 10 peptide combinations (50ug / ml), peptides using a tetramer binding assay (Tetramer binding assay) It is a graph comparing the frequency (%) of Tetramer+CD8+T cells specific to the peptide in the group treated with the combination (50ug/ml) and lipopeptide and poly(I:C) adjuvant.
Figure 4b is a graph comparing the number of Tetramer + CD8 + T cells specific to the CHA-2 peptide through the tetramer binding assay of Figure 4a.
4c shows CHA-8 (IL-2 20U/ml) peptide alone (5ug/ml or 50ug/ml), 10 peptide combinations (50ug/ml), and peptide combinations (50ug/ml) using tetramer binding analysis. ) and a graph comparing the frequency (%) of Tetramer + CD8 + T cells specific to peptides in the groups treated with lipopeptide and poly (I:C) adjuvant.
Figure 4d is a graph comparing the number of Tetramer + CD8 + T cells specific to the CHA-8 peptide through the tetramer binding assay of Figure 4c.
4e shows CHA-9 (IL-2 20U/ml) peptide alone (5ug/ml or 50ug/ml), 10 peptide combinations (50ug/ml), and peptide combinations (50ug/ml) using tetramer binding analysis. ) and a graph comparing the frequency (%) of Tetramer + CD8 + T cells specific to peptides in the groups treated with lipopeptide and poly (I:C) adjuvant.
Figure 4f is a graph comparing the number of Tetramer + CD8 + T cells specific to the CHA-9 peptide through the tetramer binding assay of Figure 4e.
4g shows CHA-15 (IL-2 20U/ml) peptide alone (5ug/ml or 50ug/ml), 10 peptide combinations (50ug/ml), and peptide combinations (50ug/ml) using tetramer binding analysis. ) and a graph comparing the frequency (%) of Tetramer + CD8 + T cells specific to peptides in the groups treated with lipopeptide and poly (I:C) adjuvant.
Figure 4h is a graph comparing the number of Tetramer + CD8 + T cells specific to the CHA-15 peptide through the tetramer binding assay of Figure 4g.
Figure 5a is a graph confirming the cytotoxic effect of the peptide combination derived from the tumor-associated antigen provided in the present invention in the colorectal cancer cell line SW620.
Figure 5b is a graph showing the results of Figure 5a by PBMC (Donor 8, 9, 10) of 3 healthy people.
Figure 5c is a graph confirming the cytotoxic effect of the peptide combination derived from the tumor-associated antigen provided in the present invention in the prostate cancer cell line PC-3.
Figure 5d is a graph showing the results of Figure 5c by PBMC (Donor 8, 9, 10) of 3 healthy people.
Figure 5e is a graph confirming the cytotoxic effect of the peptide combination derived from the tumor-associated antigen provided in the present invention in the breast cancer cell line MCF-7.
Figure 5f is a graph showing the results of Figure 5e by PBMC (Donor 8, 9, 10) of 3 healthy people.
Figure 5g is a graph confirming the cytotoxic effect (CTL activity) by the peptide combination derived from the tumor-associated antigen provided in the present invention in the ovarian cancer cell line OVCAR-3.
Figure 5h is a graph showing the results of Figure 5g by PBMC (Donor 8, 9, 10) of 3 healthy people.
6 is a graph comparing the levels of Granzyme B secretion by peptide combinations derived from tumor-associated antigens provided in the present invention in SW620, PC-3, MCF-7, and OVCAR-3 cell lines.
Figure 7a shows the cytotoxic effect of Survivin + MAGE-A3 peptide combination (S + M peptide), hTERT + WT-1 peptide (H + W peptide) combination, and 10 peptide combinations on colon cancer cell line SW620 in Donor 8 ( CTL activity) is a graph comparing ( ^# P<0.05 10 peptide combination group vs no stimulation group, ^$ P<0.05 10 peptide combination group vs H+W group, ^& P<0.05 10 peptide combination group vs S+ group M).
Figure 7b shows the cytotoxic effect of Survivin + MAGE-A3 peptide combination (S + M peptide), hTERT + WT-1 peptide (H + W peptide) combination, and 10 peptide combinations on colon cancer cell line SW620 in Donor 9 ( CTL activity) is a graph comparing ( ^# P<0.05 10 peptide combination group vs no stimulation group, ^$ P<0.05 10 peptide combination group vs H+W group, ^& P<0.05 10 peptide combination group vs S+ group M).
Figure 7c shows the cytotoxic effect of Survivin + MAGE-A3 peptide combination (S + M peptide), hTERT + WT-1 peptide (H + W peptide) combination, and 10 peptide combinations on colon cancer cell line SW620 in Donor 10 ( CTL activity) is a graph comparing ( ^# P<0.05 10 peptide combination group vs no stimulation group, ^$ P<0.05 10 peptide combination group vs H+W group, ^& P<0.05 10 peptide combination group vs S+ group M).
Figure 7d is a graph comparing the levels of Granzyme B secretion by Survivin + MAGE-A3 peptide combination (S + M peptide), hTERT + WT-1 peptide (H + W peptide) combination, and 10 peptide combinations in colorectal cancer cell line SW620. (n is the number of peptides, Donor 8-10).
Figure 8a compares the number of IFN-γ-producing cells in a test vaccine prepared with a peptide combination derived from a tumor-associated antigen provided in the present invention and a lipopeptide and a poly(I:C) adjuvant or other TLR adjuvants in a mouse model. It is a graph [from left, PBS-negative control group, peptide treatment group, peptide + TLR2 ligand treatment group, peptide + TLR3 ligand treatment group, peptide + TLR7/8 (Imiquimod) ligand treatment group, peptide + TLR9 ligand treatment group (CpG) , peptide + L-pampo treatment group].
8b is a graph comparing peptide-specific IFN-γ secretion levels in FIG. 8a.
Figure 9 is a graph comparing the number of IFN-γ producing cells in a test vaccine prepared with 10 peptide combinations alone, 10 peptide combinations and L-pampo, and 10 peptide combinations and Lipo-pam provided in the present invention in a mouse model. .

Hereinafter, the present invention will be described in detail.

The present invention provides an anti-cancer vaccine composition comprising [peptides derived from tumor-associated antigens].

The tumor associated antigen (TAA) is a protein expressed in cancer or tumor cells. Examples of TAAs are novel antigens (neoantigens expressed during tumorigenesis and altered from analogous proteins in normal or healthy cells), products of oncogenes and tumor suppressor genes, overexpressed or abnormally expressed cellular proteins (e.g., HER2, MUC-1), antigens produced by oncogenic viruses (eg EBV, HPV, HCV, HBV, HTLV), cancer testis antigens (CTA, eg MAGE family, NY-ESO) and cell type specific differentiation Antigen (eg MART-1). TAA sequences can be found experimentally or in published scientific papers, or in the Ludwig Institute for Cancer Research (www.cta.lncc.br/) database, Cancer Immunity database (cancerimmunity.org/peptide) and TANTIGEN Tumor T cell antigen database (cvc It can be found through publicly available databases such as .dfci.harvard.edu/tadb/).

On the other hand, a tumor specific antigen (TSA) is an antigen produced by a specific type of tumor that does not appear in normal cells of a tumor-generated tissue. TSA includes shared antigens, neoantigens, and unique antigens. Tumor-specific peptides are not expressed or minimally expressed in normal healthy cells or tissues, but in a high proportion (high frequency) of subjects with a specific disease or condition, such as a specific cancer type or type of cancer derived from a tissue. expressed (in cells or tissues). Alternatively, a tumor-specific peptide may be expressed at low levels in normal healthy cells, but expressed at high levels in diseased (eg, cancer) cells or subjects with a disease or condition.

The [peptides derived from tumor-associated antigens] are derived from hTERT and are derived from HLA-A02 (MHC Class I) type peptide of SEQ ID NO: 1 (SEQ ID NO: 1: ILAKFLHWL), derived from hTERT and derived from HLA-A24 (MHC Class I) Peptide of SEQ ID NO: 2 of type (SEQ ID NO: 2: VYAETKHFL), derived from hTERT and derived from HLA-DRB1 (MHC Class II) Peptide of SEQ ID NO: 3 of type (SEQ ID NO: 3: EARPALLTSRLRFIPK), derived from hTERT and derived from HLA-DRB1 ( Peptide of SEQ ID NO: 4 of MHC Class II) type (SEQ ID NO: 4: RPGLLGASVLGLDDI), derived from Survivin and HLA-A02 (MHC Class I) type of peptide SEQ ID NO: 5 (SEQ ID NO: 5: ELTLGEFLKL), from Mage-A3 The peptide of SEQ ID NO: 8 derived from HLA-A02 (MHC Class I) type (SEQ ID NO: 8: KVAELVHFL), the peptide of SEQ ID NO: 9 derived from Mage-A3 and of HLA-A24 (MHC Class I) type (SEQ ID NO: 9 : IMPKAGLLI), a peptide of SEQ ID NO: 12 derived from MUC-1 and of HLA-A02 (MHC Class I) type (SEQ ID NO: 12: STAPPVHNV), a sequence derived from WT-1 and of HLA-A02 (MHC Class I) type It may be one or more selected from the group consisting of the peptide of SEQ ID NO: 15 (SEQ ID NO: 15: RMFPNAPYL) and the peptide of SEQ ID NO: 16 (SEQ ID NO: 16: KRYFKLSHLQMHSRKH) derived from WT-1 and of HLA-DRB1 (MHC Class II) type. .

When treated with PBMCs obtained from healthy people, the immunogenicity of each peptide was higher than that of the control group not treated with the peptide, but the degree was different (FIG. 1). Based on these results, we excluded CHA-6, CHA-10, and CHA-11, which consistently showed the lowest immunogenicity. The reason why peptides showing low immunogenicity were removed is that immunotolerance may be induced when these peptides are added together. Therefore, 10 peptides (SEQ ID NOs: 1-5, 8, 9, 12, 15 and 16) were finally confirmed based on the results of immunogenicity among 13 peptides (Table 1).

Then, in order to select a peptide combination that can induce the most effective immune response against various tumor-related antigens from the 10 confirmed peptides, the peptides corresponding to each of the 5 antigens are first mixed and treated with 6 healthy human PBMCs After that, the increase or decrease in immunogenicity was analyzed through the ELISPOT test method. As a result of the experiment, when each of the five antigens was mixed, it was confirmed that the combination of hTERT and WT-1 and the combination of MAGE-A3 and the peptide corresponding to Survivin effectively enhanced immunogenicity (FIG. 2).

In order to examine whether the immunogenicity increases according to the combination of the number of peptides, the PBMCs of 3 healthy people were treated with the peptides in various combinations under various conditions, and the ELISPOT results according to the number of peptides are shown in the figure (FIG. 3). As a result of the experiment, the group treated with 10 peptide combinations showed higher immunogenicity than the group treated with other combinations, and in particular, it was confirmed that the immunogenicity was enhanced more than the hTERT + WT-1 combination and the Survivin + MAGE-A3 combination (Fig. 3).

This is because the combination of 10 peptides, namely CHA-1, CHA-2, CHA-3, CHA-4, CHA-5, CHA-8, CHA-9, CHA-12, CHA-15 and CHA-16, is It is composed of peptides derived from cancer antigens (hTERT, Survivin, MAGE-A3, MUC1, and WT-1), which can induce anti-cancer immune responses to various cancer antigens and stimulate CD8 and CD4 T cells, which are important for anti-cancer immune responses. Since it contains peptides for MHC Class I and II that can be activated together, and shows the most enhanced immunogenicity, it was confirmed that it is the most effective peptide epitope combination for treatment and prevention vaccines for various carcinomas.

The [peptides derived from tumor-associated antigens] and combinations thereof recognize MHC Class I, MHC Class II or both.

In addition, the present invention provides an anti-cancer vaccine composition comprising [a peptide derived from a tumor-associated antigen] and [an adjuvant composed of a lipopeptide and an immunoactive substance].

The lipopeptide may be composed of a fatty acid bound to a glycerol molecule and several amino acids. The number of amino acids constituting the fatty acid or lipopeptide in the glycerol molecule may be one or more. At this time, fatty acids and amino acids may be chemically modified. The lipopeptide may be a part of a molecule derived from a gram-positive or gram-negative bacterium or mycoplasma, or a lipoprotein in the form of an entire molecule.

The lipopeptides are Pam3Cys-SKKKK, Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, Pam-CSKKKK, Dhc-SKKKK And at least one selected from the group consisting of FSL-1 (Pam2CGDPKHPKSF), but is not limited thereto.

The immunoactive substance may be at least one selected from the group consisting of poly(I:C), QS21, MPLA (Monophosphoryl Lipid A), CpG, and Flagellin. The poly(I:C) has been used as a potent derivative of type 1 interferon in in vitro and in vivo studies. Furthermore, poly(I:C) is known to stably and mature dendritic cells, which are the most potent antigen-presenting cells in mammals (Rous, R. et al., International Immunol., 2004). According to these previous reports, poly(I:C) is a strong IL-12 inducer, and IL-12 is an important immunoactive substance that induces cellular immune responses and IgG2a or IgG2b antibody formation by promoting immune responses to Th1 development am.

The poly(I:C) may be 50 to 5,000 bp in length. The poly(I:C) is 10 to 150, 10 to 90, 10 to 50, 10 to 30, 30 to 60, 30 to 90, 30 to 150, 30 to 50, 10 to 1500, 10 to 900, 10 to It may be included in the adjuvant at a concentration of 500, 10 to 300, 30 to 600, 30 to 900, 30 to 1500 or 30 to 500 μg/dose, but is not limited thereto.

The QS21 is a fraction of a saponin substance called triterpene glucoside having a molecular weight of 1990.14 Da extracted from the bark of the tree Quillaja saponaria Molina of South America. QS21 is known to induce humoral and cellular immune responses by secreting Th1-type cytokines from antigen-presenting cells such as macrophages and dendritic cells when used together with lipids such as monophosphoryl lipid A (MPLA) and cholesterol.

The QS21 is 1 to 150, 1 to 90, 1 to 50, 1 to 30, 3 to 60, 3 to 90, 3 to 150, 3 to 50, 1 to 1500, 1 to 900, 1 to 500, 1 to 300 , 3 to 600, 3 to 900, 3 to 1500 or 3 to 500 μg / dose may be included in the adjuvant, but is not limited thereto.

A tetramer binding assay was performed to confirm the proliferation of CD8+ T cells. When experiments were conducted on CHA-2, CHA-8, CHA-9, and CHA-15 peptides, which are capable of tetramer synthesis, a group of 10 peptide combinations showed that each peptide was more specific than when treated alone. The number and frequency (%) of CD8+ T cells were increased, and it was confirmed that the number and frequency of CD8+ T cells were increased for each peptide by lipopeptide and poly(I:C) adjuvant (FIG. 4).

These results show that a combination of 10 selected peptides can increase CD8+ T cells more effectively than a single peptide that can activate CD8+ T cells. It shows that there is a synergistic effect. In particular, in a combination of 10 peptides, CD4+ T cells are activated by peptides (CHA-3, CHA-4, CHA-16) that can bind to MHC class II, and with the help of the activated CD4+ T cells, peptide-specific This is because more CD8+ T cells can proliferate. In addition, it can be seen that the synergistic effect of the proliferation of such CD8+ T cells was maximized due to the Th1 response induced by the known lipopeptide and poly(I:C) adjuvant (FIGS. 4a to 4h).

In order to determine whether T cells proliferated and functionally activated by a combination of peptides derived from various tumor-associated antigens can recognize and kill cancer cells as cytotoxic T cells, CTLs, that is, effector cells and cancer cells were co-cultured and then treated with CTLs. The frequency of cancer cells killed by the cells was analyzed using flow cytometry.

As a result of the experiment, the e effector cells activated by treating 3 healthy human PBMCs with the peptide combination did not contain peptides against the colorectal cancer cell line SW620, prostate cancer cell line PC-3, breast cancer cell line MCF-7, or ovarian cancer cell line OVCAR-3. It was confirmed that more cancer cells were killed than the cultured effector cells. In addition, these results were found to be cell-specific cytotoxic effects, as more cancer cells died as the number of effector cells increased (FIGS. 5a to 5h).

In addition, to confirm the activity of T cells, which are proliferated and functionally activated by peptide combinations derived from various tumor-associated antigens, against various cancer cells, Granzyme B secretion was confirmed using ELISA in culture medium in which effector cells and cancer cells were co-cultured. . Granzyme B is an enzyme secreted by CD8 T cells, especially CTL, and plays an important role in directly killing cancer cells.

As a result of the experiment, the effector cells activated by treating 6 healthy human PBMCs with a combination of peptides and colon cancer cell line SW620, prostate cancer cell line PC-3, breast cancer cell line MCF-7, or ovarian cancer cell line OVCAR-3 were cultured together. It was confirmed that Granzyme B was significantly higher than that of the culture medium in which effector cells and cancer cells were cultured together. Through this, it was found that CTL recognizes cancer cells and secretes Granzyme B, thereby inducing death of cancer cells (FIG. 6).

These results show that tumor antigen-specific T cells, which are proliferated and functionally activated by the peptide combination, can recognize cancer cells as cytotoxic T cells and secrete Granzyme B to directly kill cancer cells.

Next, the 10 peptide combinations confirmed to exhibit excellent immunogenicity through FIGS. 3, 4, and 5 were more immunogenic than the Survivin + MAGE-A3 or hTERT + WT-1 peptide combinations that showed high immunogenicity in FIG. 2 High CTL activity efficacy was compared.

When cytotoxicity and Granzyme B secretion were analyzed for colon cancer cell line SW620, 3 healthy human PBMCs were treated with 10 peptide combinations, and activated effector cells were treated with Survivin+MAGE-A3 or hTERT+WT-1 peptide combinations. It was confirmed that significantly higher Granzyme B secretion and more cancer cells could be killed than activated effector cells (FIGS. 7a to 7d).

This is because the selected 10 peptide combinations are composed of peptides derived from various tumor-associated antigens (hTERT, Survivin, MAGE-A3, MUC-1 and WT-1), which can induce anti-cancer immune responses against various cancer antigens. , it shows that since CD8 and CD4 T cells, which are important for anti-cancer immune response, can be activated together, the immune response against cancer cells is highly induced and more cancer cells can be killed by activated CTL.

Finally, in the mouse model, induced by test vaccines prepared to include 10 peptide combinations, lipopeptide and poly(I:C) adjuvant, and test vaccines prepared to include 10 peptide combinations and other TLR adjuvants. Cellular immune responses were compared. The experimental groups were PBS negative control, 10 peptide combinations, 10 peptides and TLR2 ligand, 10 peptides and TLR3 ligand, 10 peptides and TLR7/8 ligand (Imiquimode), 10 peptides and TLR9 ligand (CpG), and 10 peptides. and L-pampo (lipopeptide and poly (I:C) adjuvant) in a total of 7 groups (FIGS. 8a to 8b). As a result of the experiment, the number of IFN-γ-producing cells in the test vaccine using lipopeptide and poly(I:C) adjuvant, that is, L-pampo, was significantly higher than that of the test vaccine using TLR2, TLR3, TLR7/8 or TLR9 adjuvant. It was confirmed that the secretion of each peptide-specific IFN-γ was also increased in the test vaccine using L-pampo than the test vaccine using TLR2, TLR3, TLR7/8 or TLR9 adjuvant (FIGS. 8a to 8b) .

In addition, in the mouse model performed in FIG. 8, the test vaccine prepared with Lipo-pam adjuvant composed of liposomes and immunoactive substances with 10 peptide combinations and lipopeptides inserted on the surface induces a cellular immune response similar to L-pampo As a result, it was confirmed that the test vaccine using Lipo-pam significantly increased the number of cells producing IFN-γ compared to the control group, similar to the vaccine using L-pampo (FIG. 9).

Taking these results together, the anti-cancer vaccine composition provided in one aspect of the present invention is a cellular immune response capable of killing cancer cells compared to vaccine compositions containing other adjuvants, such as TLR2, TLR3, TLR7/8 or TLR9 adjuvants. It shows that it is an anti-cancer vaccine composition that can strongly induce.

On the other hand, in one embodiment of the present invention, the anti-cancer vaccine composition is prepared by mixing the adjuvant with the lipopeptide and the immunoactive substance at a certain ratio.

The formulation of the anti-cancer vaccine composition may be an oil emulsion or an aqueous formulation.

The anti-cancer vaccine composition may additionally include a buffer, an isotonic agent, a preservative, a stabilizer and a solubilizing agent. Phosphate, acetate, ammonium phosphate, ammonium carbonate, citrate and the like can be used as the buffering agent.

In the composition ratio of the entire vaccine composition including the above composition, in the lipopeptide and poly(I:C) adjuvant, the lipopeptide is preferably included in a weight ratio of 0.01 to 0.5% by weight, but in a weight ratio of 0.01 to 0.1% by weight. It is most preferably included, and poly(I:C) is preferably included in a weight ratio of 0.01 to 0.4% by weight, and most preferably included in a weight ratio of 0.01 to 0.08% by weight, but is not limited thereto.

In addition, as the isotonic agent, sodium chloride is preferably included in a weight ratio of 0.1 to 0.8% by weight, more preferably included in a weight ratio of 0.2 to 0.6% by weight, and according to a preferred embodiment of the present invention, a weight ratio of 0.44% by weight. It is most preferable to include as.

In addition, as the buffering agent, phosphate, acetate, ammonium phosphate, ammonium carbonate, citrate, etc. are preferably used, and according to a preferred embodiment of the present invention, phosphate and acetate are more preferably used, but are not limited thereto. The phosphate is preferably included in a weight ratio of 0.01 to 0.4% by weight, and more preferably included in a weight ratio of 0.01 to 0.2% by weight, but according to a preferred embodiment of the present invention, it is most preferably included in a weight ratio of 0.13% by weight. desirable. In addition, the acetate is preferably included in a weight ratio of 0.01 to 0.3% by weight, and more preferably included in a weight ratio of 0.01 to 0.2% by weight, but according to a preferred embodiment of the present invention, it is included in a weight ratio of 0.08% by weight is most preferable Therefore, the buffering agent in the vaccine composition of the present invention is preferably included in a weight ratio of 0.02 to 0.6% by weight, but according to a preferred embodiment of the present invention, it is most preferably included in a weight ratio of 0.15 to 0.25% by weight.

Meanwhile, in another embodiment of the present invention, the adjuvant is prepared by mixing a liposome having a lipopeptide inserted on its surface and an immunoactive substance.

The lipopeptide is added to the liposome at a concentration of 20 to 250, 20 to 50, 50 to 250, 150 to 250, 50 to 150, 20 to 2500, 20 to 500, 50 to 2500, 150 to 2500 or 50 to 1500 μg/dose. can be inserted.

The liposomes are composed of lipids.

The lipid may be a cationic, anionic or neutral lipid. For example, DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DDA (Dimethyldioctadecylammonium), DC-chol (3β-[N-( N',N'-Dimethylaminoethane)-carbamoyl]cholesterol), DOPG (1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)]), DPPC (1,2-dipalmitoyl-sn -glycero-3phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and at least one selected from the group consisting of cholesterol, but is not limited thereto.

The lipid is 15 to 300, 15 to 150, 15 to 90, 15 to 50, 15 to 40, 20 to 30, 15 to 3000, 15 to 1500, 15 to 900, 15 to 500, 15 to 400 or 20 to 300 It can be incorporated into liposomes at a concentration of μg/dose.

The adjuvant is composed of a positively charged liposome in which a positively charged lipopeptide is inserted into a lipid bilayer and a negatively charged immunoactive substance.

The anti-cancer vaccine composition can induce both a humoral immune response and a cellular immune response.

The anti-cancer vaccine composition can enhance the Th1 immune response. An antibody of IgG2a or IgG2b, which enhances the Th1 immune response effective for antiviral and anticancer immune responses, is produced by cytokines produced by helper T cell 1 (Th1). Therefore, the anti-cancer vaccine composition of the present invention can also be used as a preventive or therapeutic agent for cancer progressing from viral infection.

The humoral immune response can be induced from secretion of cytokines by increasing T follicular helper cells (Tfh) by the adjuvant of the present invention.

The cellular immune response can be induced from increased secretion of IFN-γ, TNF-α and granzyme B.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the anti-cancer vaccine composition of the present invention as an active ingredient.

The anti-cancer vaccine composition may have characteristics as described above.

In one embodiment of the present invention, the anti-cancer vaccine composition may include an adjuvant and an antigen. The adjuvant may include a liposome into which a lipopeptide is inserted, and may further include an immunoactive substance thereto.

The prophylactic or therapeutic agent of the present invention may contain a pharmaceutically acceptable carrier, and may be formulated for human or veterinary use and administered through various routes. The route of administration may be oral, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal transdermal, transnasal or the like route. Preferably, it is formulated and administered as an injection. Injections are aqueous solvents such as saline solution and intravenous solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (eg, oleic acid ethyl, etc.), alcohols (eg, ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) Stabilizers to prevent deterioration (e.g., ascorbic acid, sodium hydrogensulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, and inhibition of microbial growth Preservatives (for example, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

The pharmaceutical composition for preventing or treating cancer of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount that can exhibit a vaccine effect and does not cause side effects or serious or excessive immune reactions at the same time, and the exact dosage concentration may vary depending on the antigen to be included in the vaccine. In addition, the preventive or therapeutic agent can be easily determined by a person skilled in the art according to factors well known in the medical field, such as age, weight, health, sex and sensitivity to drugs, administration routes and administration methods of patients. The administration may be one to several administrations.

The cancers include ovarian cancer, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, intracranial race, ependymoma, brainstem tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus cancer, nasopharyngeal cancer, salivary gland cancer, Hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinum tumor, esophageal cancer, breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer , bladder cancer, kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, uterine sarcoma, vaginal cancer, female external genital cancer, and skin cancer, but is not limited thereto.

The anti-cancer vaccine composition can be used in combination with one or more treatments selected from the group consisting of chemo-cancer agents, targeted anti-cancer agents, immune checkpoint inhibitors, and radiation therapy, and exhibits a synergistic anti-cancer effect compared to existing therapies.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following examples and experimental examples are only to illustrate the present invention, and the content of the present invention is not limited thereto.

<Example 1> Discovery of MHC class I and MHC class II antigen peptide epitopes derived from tumor-associated antigens

Example 1-1. Selection of peptide candidates for inducing immunogenicity

In order to select candidates for peptides to induce immunogenicity, Peptides derived from known tumor-associated antigens, which are undergoing clinical trials for various carcinomas, were selected as candidates. The selected tumor-associated antigens were hTERT, Survivin, MAGE-A3, MUC-1, EGFRvIII, NY-ESO-1 and WT-1 antigens, and 16 peptides derived from these antigens were selected (Table 1).

division tumor-associated antigen amino acid sequence HLA classes HLA type sequence number CHA-1 hTERT ILAKFLHWL (540-548) I A02 One CHA-2 hTERT VYAETKHFL (324-332) I A24 2 CHA-3 hTERT EARPALLTSRLRFIPK (611-626) II DRB1 3 CHA-4 hTERT RPGLLGASVLGLDDI (672-686) II DRB1 4 CHA-5 Survivin ELTLGEFLKL (96-104) I A02 5 CHA-6 Survivin LMLGEFLKL (97-104) I A02 6 CHA-7 Survivin AYACNTSTL (80-88) I A24 7 CHA-8 Mage-A3 KVAELVHFL (112-120) I A02 8 CHA-9 Mage-A3 IMPKAGLLI (195-203) I A24 9 CHA-10 Mage-A3 TQHFVQENYLEY (246-260) II DRB1 10 CHA-11 Mage-A3 FLWGPRALV (271-279) I A02 11 CHA-12 MUC-1 STAPPVHNV (179-187) I A02 12 CHA-13 EGFRvIII LEEKKGNYVVTDHC (1-13) II DRB1 13 CHA-14 NY-EOS-1 SLLMWITQCFLPVF (157-170) II DRB1 14 CHA-15 WT-1 RMFPNAPYL (126-134) I A02 15 CHA-16 WT-1 KRYFKLSHLQMHSRKH (332-346) II DRB1 16

Among these 16 peptides, 3 peptides (CHA-7, CHA-13, CHA-14) all have cysteine at amino acid residues, which prevents them from binding to MHC molecules by forming disulfide bonds. It was excluded because it could be disruptive.

An in silico algorithm was used to determine whether the 13 peptides thus selected had good binding affinity to MHC class I or MHC class II.

As a result, by confirming that the selected 13 peptides originate from various tumor-associated antigens and specifically bind to HLA-A02/A24 and HLA-DRB1, peptide candidates for inducing immunogenicity of the 13 peptides confirmed as The confirmed 13 peptide sequences were synthesized into peptides by requesting a peptide synthesis company (Anigen, Korea).

Example 1-2. Confirmation of immunogenicity of selected peptides and selection of final peptides

In order to confirm the immunogenicity of the 13 peptides selected in Example 1, an interferon gamma (IFNg)-fixed enzyme antibody method (Enzyme Linked Immuno-SPOT assay, ELISPOT) was performed.

This experiment is a sensitive immunoassay that can detect cytokines secreted by only one cell. By attaching cytokines secreted when cells are activated to capture antibodies, proteins or proteins expressed on the cell surface It is possible to measure secreted cytokines without being affected by degrading enzymes. After attaching the cytokine to the capture antibody, removing the cells, adding a detecting antibody coupled to an enzyme capable of recognizing the attached capture antibody, and then adding a chromogenic substrate for this enzyme again to secrete the cytokine. It is a method in which a color spot is formed at the location of the cell being stimulated and the number of cells reacting to the stimulating antigen is confirmed by the number of colored spots.

Peripheral blood mononuclear cells (PBMC) required for the experiment were isolated from human venous blood as follows. After diluting the venous blood with 1X PBS, it was placed in a SepMate-50 tube and centrifuged at 800 g for 20 minutes at room temperature. After centrifugation, plasma and separated PBMCs are washed, and then put into each well of a 6 well tissue culture plate at a concentration of 1 x 10 ⁶ cells/mL, and then placed in an incubator ( incubator) overnight (37 ℃, 5% CO ₂ ). The next day, after recovering the PBMC culture medium, it was centrifuged at 2000 rpm for 15 minutes at room temperature. After centrifugation, the PBMCs were seeded in a 6 well cell culture plate, and cultured with peptides to stimulate stimulation.

At this time, the 13 peptides selected in Example 1 were treated alone or in combination with various peptides, and an untreated group and a ConA group were also prepared for comparative analysis. After culturing in a 6-well cell culture plate, 200 μL of the PBMC cell culture medium was dispensed into each well of the ELISPOT plate and cultured for 24 hours at 37 °C and 5% CO ₂ conditions. After incubation, ELISPOT analysis was prepared by washing 5 times with 1 X PBS. The detection antibody (R4-642-biotin) was diluted in PBS (0.5% FBS) at 100 μL/well and reacted at room temperature for 2 hours. Then, after washing 5 times with 1 X PBS, Streptavidin-HRP was diluted in PBS (0.5% FBS), added at 100 μL/well, and allowed to react for 1 hour at room temperature. After that, they were washed with 1 X PBS, and 100 μL/well of TMB substrate solution was added. When the color development of the spot was clearly raised, the reaction was terminated by washing with DW 6 times. After drying, they were wrapped in foil and dried in the shade for one day. After attaching ELI-FOIL to the dried ELISPOT plate to print a membrane, the number of spots in each well was read on an ELISPOT reader. From the number of spots in the test group, the number of spots in the untreated group was plotted as a limiting value.

As a result, as shown in FIG. 1, it was confirmed that immunogenicity was increased in all 13 peptide-treated groups compared to the untreated control group. Among them, CHA-6, CHA-10, and CHA-11, which consistently showed the lowest immunogenicity, were excluded. This is because, when a peptide showing low immunogenicity is added together, immune tolerance may be induced due to such a peptide. Therefore, based on the results of immunogenicity among 13 peptides, 10 peptides (CHA-1 to CHA-5, CHA-8, CHA-9, CHA-12, CHA-15 and CHA-16) were finally confirmed. (Table 1).

Afterwards, in order to select a peptide combination that can induce the most effective immune response against various cancer antigens from the 10 confirmed peptides, the peptides corresponding to each of the 5 antigens were first mixed and treated with 6 healthy human PBMCs. Afterwards, the increase or decrease in immunogenicity was analyzed through the ELISPOT test method.

As a result, as shown in FIG. 2, the combination of hTERT and WT-1 and the peptides corresponding to MAGE-A3 and Survivin more effectively enhances immunogenicity compared to the combination of five antigens mixed for each antigen. Confirmed. In addition, when a combination of other antigens was further mixed with this combination or when other antigens other than these two combinations were combined, it was confirmed that even if the immunogenicity was reduced or increased, it did not increase more than the value of these two combinations.

In the above experiment, there were 6 peptides included in the combination of hTERT and WT-1 antigens (4 derived from hTERT and 2 derived from WT-1), and 3 peptides included in the combination of MAGE-A3 and Survivin (MAGE-1 derived). 32 A and 1 Survivin), an experiment was performed to determine whether immunogenicity increases according to the combination of the number of peptides.

To this end, as shown in FIG. 3, the peptides were combined under various conditions and treated with three healthy human PBMCs, and the ELISPOT results according to the number of peptides were confirmed.

As a result, as shown in FIG. 3, the group treated with 10 peptides showed higher immunogenicity than the other combination groups, and in particular, it was confirmed that the immunogenicity was enhanced more than the hTERT + WT-1 combination and the Survivin + MAGE-A3 combination. .

The above results show that 10 peptide combinations, namely CHA-1, CHA-2, CHA-3, CHA-4, CHA-5, CHA-8, CHA-9, CHA-12, CHA-15 and CHA-16 Since the combination is composed of peptides derived from various cancer antigens (hTERT, Survivin, MAGE-A3, MUC-1 and WT-1), it can induce anti-cancer immune responses to various cancer antigens, and CD8, which is important for anti-cancer immune responses It is thought to show the most enhanced immunogenicity because it contains both MHC Class I and II peptides that can activate CD4 T cells together. Therefore, it was confirmed that the 10 peptide combinations are the most effective peptide epitope combinations for therapeutic and preventive vaccines against various carcinomas.

<Example 2> Preparation of anti-cancer vaccine containing peptides derived from tumor-associated antigens

All peptides were synthesized by standard and well-established solid-phase peptide synthesis methods using Fmoc chemistry using appropriate resins from the C-terminus to the N-terminus (Anigen, Korea). Purification and purity of each individual peptide was confirmed by mass spectrometry and HPLC analysis. The peptide was obtained as a white lyophilized compound and >95% pure. All peptides were used at a concentration of 10 mg/ml.

<Example 3> Preparation of anti-cancer vaccine containing [peptide derived from tumor-associated antigen] and [adjuvant composed of lipopeptide and immunoactive substance]

Example 3-1. L-pampo

An anti-cancer vaccine was prepared by mixing the peptide combination selected in Example 1-2 of the present invention with Pam3Cys-SKKKK and poly(I:C) adjuvant.

First, in order to observe the adjuvant effect when formulated using Pam3Cys-SKKKK and poly(I:C) simultaneously, a test vaccine in which Pam3Cys-SKKKK and poly(I:C) were mixed was prepared. When preparing the vaccine mixture containing the peptide, a 10X buffer solution (pH 7.0) containing 150 mM NaCl and 10 mM sodium phosphate was used.

Example 3-2. Lipo-pam

First, DOTAP:DPPC liposomes were prepared. To this end, DOTAP and After each DPPC was melted, the organic solvent was vaporized with nitrogen gas while turning so that the mixed solution of DOTAP and DPPC could be evenly distributed on the wall of the glass container. At this time, a thin film was formed on the wall, and the organic solvent remaining in the formed film was removed by storing it in a vacuum desiccator. Distilled water was added to the completely dried lipid film to rehydrate it. When a multilamella vesicle (MLV) suspension solution was created, a 2X buffer solution (pH 7.0) containing 300 mM NaCl in 20 mM sodium phosphate was added in the same amount as distilled water. The resulting MLV was sonicated for 5 cycles of 5 minutes at 3 sec/3 sec (pulse on/off) to prepare DOTAP:DPPC liposomes in the form of small unilamellar vesicles (SUVs).

Subsequently, Lipo-pam was prepared in the same manner as the DOTAP:DPPC liposome, except that DOTAP, DPPC, and Pam3-CSKKKK were dissolved in an organic solvent, and then DOTAP and DPPC were mixed and Pam3-CSKKKK was added thereto. did A vaccine mixture is prepared by adding an immunoactive substance adjuvant ((poly(I:C)) and a peptide to this Lipo-pam, and at this time, a 10X buffer solution (pH 7.0) containing 150 mM NaCl and 10 mM sodium phosphate is prepared. was used.

<Experimental Example 1> Verification of CD8 T cell-specific immunogenicity induction of peptide combinations

CD8+ T cells, as the most important immune cells in anticancer immunity, directly kill cancer cells and also prevent the proliferation of cancer cells. Therefore, when there are many CD8+ T cells specific for tumor-associated antigens, more effective anticancer immunity can be induced. In Experimental Example 1, tetramer binding analysis was performed to confirm the proliferation of CD8+ T cells by the combined administration of peptide single, peptide combination and peptide combination and adjuvant (lipopeptide and poly (I: C)) presented in Example 1 (tramer binding assay) was performed.

In the tetramer binding assay, after making 4 MHC/peptide complexes into one complex, fluorophore-labeled streptavidin is attached to the “MHC tetramer” to perform flow cytometry analysis. ) to measure the number or frequency (%) of MHC tetramer-bound CD8+ T cells, thereby sensitively verifying the antigen-specific CD8 T cell response, especially the CTL (cytotoxic T lymphocyte) response.

Therefore, in this experiment, the following four tetramers were prepared using peptides for MHC class I type that can recognize CD8 T cells, that is, HLA-A02 and HLA-24 (CHA-2/HLA-A24 tetramer tetramer, CHA-8/HLA-A02 tetramer, CHA-9/HLA-A24 tetramer, and CHA-15/HLA-A02 tetramer).

In the same way as in the ELISPOT assay, PBMCs were isolated from human venous blood and dispensed by putting 200 mL into each well of a 96-well cell culture plate. The divided PBMCs were stimulated by treatment with peptides. At this time, each peptide was treated at 5 mg/mL or 50 mg/mL, and even when the 10 peptide combinations confirmed in Example 1-2 were mixed and treated, they were treated at the same concentration. At this time, a control group that was not treated with the peptide or treated with a non-specific tetramer was also prepared for analysis.

A 96-well cell culture play was placed in an incubator and cultured for 24 hours at 37 °C and 5% CO ₂ conditions. When the culture was completed, the 96-well plate was taken out, and the cultured cells were harvested into a Fluorescence Activated Cell Sorter (FACS) tube, followed by centrifugation at room temperature at 2000 rpm for 15 minutes. After centrifugation was completed, after removing the supernatant, FVS (Fixable Viability Stain) was treated and the cells were redispersed (cell resuspension). After resuspension, the FACS tube was covered with foil and reacted at room temperature for 15 minutes. After washing with 2 mL PBS, centrifugation was performed at 2000 rpm, RT, 5 minutes. After centrifugation was completed, the FACS tube was taken out, the supernatant was removed, 1 mL of FACS buffer was washed, and then placed in a centrifuge and centrifuged at room temperature at 2000 rpm for 5 minutes. After centrifugation was completed, the FACS tube was taken out, the supernatant was removed, 100 μL of FACS buffer was added, and redispersed. After redispersion, Human Fc R blocking solution and MHC tetramer were added to a FACS tube and mixed well. Wrap the FACS tube in foil and allow to react for 20 minutes at room temperature. After 20 minutes, anti-human CD3, CD45, and CD8 antibodies were added and mixed well. After wrapping the FACS tube in foil and reacting in a refrigerator, the FACS tube was taken out of the refrigerator, FACS buffer solution was added, and centrifugation was performed at room temperature. After centrifugation is complete, take out the FACS tube, remove the supernatant, add FACS buffer to resuspend the cells, and measure the number of MHC Tetramer ⁺ CD8 ⁺ T cells and MHC in total CD8 + T cells using a flow cytometer. The frequency (%) of Tetramer ⁺ CD8 ⁺ T cells was analyzed.

As a result, as shown in Figures 4a to 4h, the group of 10 peptide combinations than the case of CHA-2, CHA-8, CHA-9 and CHA-15 peptides alone, each peptide specific The frequency (%) and total number (No.) of Tetramer + CD8 + T cells increased (blue box), and when lipopeptide and poly(I:C) adjuvant were mixed with 10 peptide combinations and treated, each It was confirmed that the frequency and number of Tetramer + CD8 + T cells for the peptide increased more than when only 10 peptide combinations were added (red box).

The above results show that a combination of 10 selected peptides can increase CD8+ T cells more effectively than a single peptide that can activate CD8+ T cells, especially when the combination of 10 peptides and an adjuvant are administered together. show that there is a positive effect. In particular, CD4+ T cells are activated by peptides (CHA-3, CHA-4, CHA-16) that can bind to MHC class II in a combination of 10 peptides, and with the help of the activated CD4+ T cells, peptide-specific This is because CD8+ T cells can proliferate more. In addition, it can be seen that the synergistic effect of the proliferation of such CD8+ T cells was maximized due to the Th1 response induced by the known lipopeptide and poly(I:C) adjuvant.

<Experimental Example 2> Verification of cytotoxic ability of CD8+ T cells induced by peptide combination

In order to confirm that T cells proliferated and functionally activated by a combination of peptides derived from various tumor-associated antigens can recognize and kill cancer cells as CTLs (cytotoxic T lymphocytes), effector cells After co-cultivation with cancer cells, a cytotoxicity assay was performed to analyze the frequency of cancer cells killed by CTL using a flow cytometer.

First, in order to prepare effector cells, that is, CTL proliferated and activated by peptides, PBMCs were isolated from human venous blood, and then 10 peptides selected in Examples 1-2 were added at 10 mg/mL and 5 ng of hIL-2, respectively. /mL was added and cultured. As a control, cells cultured with only 5 ng/mL of hIL-2 without adding any peptide were used.

In order to confirm that the effector cells increased by the peptide can recognize and kill the target cancer cells specifically by MHC, HLA-A02, HLA-A24 alone or expressing both HLA-A02 and HLA-A24 Cultured with cancer cells. As culture conditions, the ratio of effector cells and target cells (cancer cells) cultured with or without the peptide combination was 2.5: 1, 5: 1, and 10: 1, respectively, and incubated together for 16 hours, and cancer cells were killed by the effector cells. The degree of death was measured using the CFSE/7-AAD (Carboxyfluorescein succinimidyl ester/7-Aminoactinomycin D) staining method.

The CFSE/7-AAD staining method stains target cancer cells with a substance called CFSE, which turns the cancer cells green. After culturing the target cancer cells stained with CFSE for 16 hours with effector cells, they are stained again with a substance called 7-AAD. Since 7-AAD stains only dead cells and displays them in red, target cancer cells expressing CFSE and 7-AAD together are identified as dead cells by effector cells. After staining, the frequency of CFSE ⁺ 7-AAD ⁺ cancer cells was measured using a flow cytometer, and the results were graphed.

Colon cancer cell line SW620, which expresses both HLA-A02 and HLA-A24; prostate cancer cell line PC-3, which expresses only HLA-24; breast cancer cell line MCF-7, which expresses only HLA-02; and ovarian cancer. As a result of each experiment targeting the cell line OVCAR-3, the effector cells activated by the peptide combination killed more cancer cells than the effector cells cultured without adding the peptide, and it was confirmed that the same trend appeared in 3 donors. did In addition, as the number of effector cells increased, more cancer cells died, confirming that the effect was a cell-specific cytotoxic effect (FIGS. 5a to 5h).

<Experimental Example 3> Confirmation of induction of Granzyme B secretion by peptide combination

Effectors to determine whether T cells proliferated and functionally activated by a combination of peptides derived from various tumor-associated antigens can secrete Granzyme B, which can directly kill cancer cells as CTL (cytotoxic T lymphocytes) The level of Granzyme B in the culture medium in which cells and cancer cells were co-cultured was analyzed using ELISA.

In the same manner as in <Experimental Example 2>, the ratios of effector cells and target cells (cancer cells) cultured with or without the peptide combination were added at 2.5: 1, 5: 1, and 10: 1, respectively, and cultured together for 16 hours. After that, the culture medium was obtained. The obtained culture medium was centrifuged at 2000 rpm for 5 minutes, and the supernatant was obtained and used for Granzyme B analysis.

Granzyme B ELISA was performed according to the corresponding protocol using MABTECH's Human Granzyme B (HRP) (cat no. 3486-1H-6) kit. 2 μg/mL of capture mAb MT34B6 was reacted at 100 μL/well in a 96-well immunoplate overnight at 4°C. After the reaction, the supernatant was removed, and 200 μL/well of PBST (PBS+0.05% Tween 20) containing 0.1% BSA was added and reacted at room temperature for 1 hour. After washing 5 times with PBST (PBS+0.05% Tween 20), each supernatant obtained or 0 - 2000 pg/mL standard material in the kit was added at 100 μL/well and reacted at room temperature for 2 hours. After washing 5 times with PBST (PBS+0.05% Tween 20), 1 μg/mL MT28-biotin was added at 100 μL/well and reacted at room temperature for 1 hour. After washing 5 times with PBST (PBS+0.05% Tween 20), 100 μL/well of Streptavidin-HRP was added and reacted at room temperature for 1 hour. Washed 5 times with PBST (PBS+0.05% Tween 20). After adding 100 μL/well of TMB substrate and reacting at room temperature for 15 minutes, 50 μL/well of 0.2M sulfuric acid was added to stop the TMB substrate reaction. After measuring the O.D value at a wavelength of 450 nm using an ELISA reader, the amount of Granzyme B in the supernatant was analyzed by comparison with a standard material.

As a result, as shown in FIG. 6, PBMCs derived from six healthy people were treated with a peptide combination to activate effector cells and colon cancer cell line SW620, prostate cancer cell line PC-3, breast cancer cell line MCF-7, or ovarian cancer cell line OVCAR. It was confirmed that significantly higher Granzyme B was measured in the culture medium incubated with -3 than in the culture medium in which effector cells and cancer cells were co-cultured without peptide. Through this, it was found that CTL recognizes cancer cells and induces the death of cancer cells by secreting Granzyme B.

<Experimental Example 4> Verification of cancer cell killing ability of cytotoxic T cells according to cancer antigen or combination of peptide numbers

Comparison of CTL activity efficacy between the 10 peptide combinations, which were confirmed to exhibit excellent immunogenicity through the above (Fig. 3), and the Survivin + MAGE-A3 or hTERT + WT-1 peptide combination, which showed high immunogenicity in the above (Fig. 2) did

The experimental method was the same as in <Experimental Examples 2 and 3>, and the colon cancer cell line SW620 was used as the cancer cell.

As a result, as shown in FIGS. 7A to 7D , effector cells activated by treating PBMCs derived from three healthy people with 10 peptide combinations were treated with Survivin + MAGE-A3 or hTERT + WT-1 peptide combinations. It was confirmed that it secreted much higher levels of Granzyme B than activated effector cells and could kill many cancer cells.

The above results show that the 10 peptide combinations are composed of peptides derived from various cancer antigens (hTERT, Survivin, MAGE-A3, MUC-1 and WT-1), which can induce anti-cancer immune responses against various cancer antigens. , CD8 and CD4 T cells, which are important for the anticancer immune response, can be activated together, suggesting that the immune response against cancer cells is highly induced and more cancer cells can be killed by activated CTLs through the induced immune response.

<Experimental Example 5> Evaluation of cellular immune response inducing efficacy of a test vaccine prepared with a peptide combination derived from a tumor-associated antigen provided in the present invention, a lipopeptide, an immunoactive material adjuvant or other TLR adjuvants in a mouse model

In the mouse model, the cellular immune response induction level of the 10 peptide combinations and the test vaccine (L-pampo) prepared to include lipopeptide and immunoactive material adjuvant or the test vaccine prepared to include other TLR adjuvants was compared. .

For the test vaccine, 50 μg each of 10 peptides were mixed, and then lipopeptide (Pam3Cys-SKKKK) and immunoactive substance (poly (I:C)) adjuvant (L-pampo) or other TLR adjuvant (TLR2) were added to the mixture. It was prepared to include lipopeptide as a ligand, poly (I:C) as a TLR3 ligand, lmiquimod as a TLR7/8 ligand, and CpG as a TLR9 ligand. Each test vaccine was subcutaneously injected three times at 1-week intervals into C57BL/6 mice at 100 μL. One week after the 3rd dose of immunization, the spleen was removed from the mouse and all splenocytes were isolated, and immunogenicity was analyzed using the ELISPOT method.

As a result, as shown in Figures 8a and 8b, in the test vaccine (L-pampo) using Pam3Cys-SKKKK and poly (I:C) adjuvant, the test vaccine using TLR2, TLR3, TLR7/8 or TLR9 adjuvant. In comparison, it was confirmed that the number of cells producing IFN-γ increased significantly, and the secretion of IFN-γ specific to each peptide also increased.

The above results show that the anti-cancer vaccine composition provided in one aspect of the present invention has a more powerful cellular immune response capable of killing cancer cells than vaccine compositions containing other adjuvants TLR2, TLR3, TLR7/8 or TLR9 adjuvants. show that it can be induced.

<Experimental Example 6> Lipo-pam adjuvant composed of an adjuvant composed of a peptide combination derived from the tumor-associated antigen provided in the present invention, a lipopeptide, and an immunoactive substance or a liposome having a lipopeptide inserted into the surface and an immunoactive substance in a mouse model Comparative evaluation of cellular immune response-inducing efficacy of test vaccines made with bunt

In the mouse model, a test vaccine (Lipo-pam) prepared to contain 10 peptide combinations, lipopeptides and immunoactive substance adjuvant, and 10 peptide combinations and lipopeptides inserted into the surface of liposomes and immunoactive substances. The induction of cellular immune responses of the test vaccines prepared with the formulated Lipo-pam adjuvant was compared.

For the test vaccine, 50 μg each of 10 peptides were mixed, and then lipopeptide (Pam3Cys-SKKKK) and immunoactive substance (poly (I:C)) adjuvant or lipopeptide (Pam3Cys-SKKKK) were inserted into the surface of the mixture. It was prepared to include Lipo-pam adjuvant composed of liposomes and immunoactive substances (poly(I:C)). Each test vaccine was subcutaneously injected three times at 1-week intervals into C57BL/6 mice at 100 μL. One week after the 3rd dose of immunization, the spleen was removed from the mouse and all splenocytes were isolated, and immunogenicity was analyzed using the ELISPOT method.

As a result, as shown in FIG. 9, the test vaccine using the Lipo-pam adjuvant composed of liposomes having Pam3Cys-SKKKK inserted on the surface and poly(I:C) was Pam3Cys-SKKKK and poly(I:C) adjuvant. Similar to the test vaccine using (Lipo-pam), it was confirmed that the number of cells producing IFN-γ increased significantly compared to the control group.

The above results show that the lipopeptide, which is an anti-cancer vaccine composition provided in one aspect of the present invention, and the immunoactive substance adjuvant or Lipo-pam adjuvant composed of liposomes and immunoactive substances inserted into the surface of the lipopeptide can kill cancer cells. It shows that it is an anti-cancer vaccine composition capable of strongly inducing a cellular immune response.

Claims (22)

An anti-cancer vaccine composition comprising [a peptide derived from a tumor associated antigen (TAA)].
An anti-cancer vaccine composition comprising [peptide derived from tumor associated antigen (TAA)] and [adjuvant composed of lipopeptide and immunoactive substance].
According to claim 1 or 2,
The tumor-associated antigen is an anti-cancer vaccine composition, characterized in that at least one selected from the group consisting of hTERT, MAGE-A3, MUC-1, Survivin and WT-1.
According to claim 1 or 2,
The anti-cancer vaccine composition, characterized in that the [peptide derived from the tumor-associated antigen] comprises any one or more of the following peptides:
i) the peptide of SEQ ID NO: 1 (SEQ ID NO: 1: ILAKFLHWL), characterized in that it is derived from hTERT and is of the HLA-A02 (MHC Class I) type;
ii) the peptide of SEQ ID NO: 2 (SEQ ID NO: 2: VYAETKHFL), which is derived from hTERT and is of the HLA-A24 (MHC Class I) type;
iii) the peptide of SEQ ID NO: 3 (SEQ ID NO: 3: EARPALLTSRLRFIPK), characterized in that it is derived from hTERT and is of the HLA-DRB1 (MHC Class II) type;
iv) the peptide of SEQ ID NO: 4 (SEQ ID NO: 4: RPGLLGASVLGLDDI), which is derived from hTERT and is of the HLA-DRB1 (MHC Class II) type;
v) the peptide of SEQ ID NO: 5 (SEQ ID NO: 5: ELTLGEFLKL), which is derived from Survivin and is of the HLA-A02 (MHC Class I) type;
vi) the peptide of SEQ ID NO: 8 (SEQ ID NO: 8: KVAELVHFL), which is derived from Mage-A3 and is of the HLA-A02 (MHC Class I) type;
vii) the peptide of SEQ ID NO: 9 (SEQ ID NO: 9: IMPKAGLLI), which is derived from Mage-A3 and is of the HLA-A24 (MHC Class I) type;
viii) the peptide of SEQ ID NO: 12 (SEQ ID NO: 12: STAPPVHNV), characterized in that it is derived from MUC-1 and is of the HLA-A02 (MHC Class I) type;
ix) the peptide of SEQ ID NO: 15 (SEQ ID NO: 15: RMFPNAPYL), which is derived from WT-1 and is of the HLA-A02 (MHC Class I) type; and
x) The peptide of SEQ ID NO: 16 (SEQ ID NO: 16: KRYFKLSHLQMHSRKH), characterized in that it is derived from WT-1 and is of the HLA-DRB1 (MHC Class II) type.
According to claim 1 or 2,
The anti-cancer vaccine composition, characterized in that the [peptides derived from tumor-associated antigens] recognize MHC Class I, MHC Class II or both.
According to claim 2
The adjuvant is an anti-cancer vaccine composition, characterized in that prepared by mixing a lipopeptide and an immunoactive substance.
According to claim 2,
The adjuvant is an anti-cancer vaccine composition, characterized in that it comprises a liposome (liposome) is inserted into the surface of the lipopeptide.
According to claim 2,
The lipopeptides are Pam3Cys-SKKKK, Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2CysSKKKK, PamDhc-SKKKK, PamCSKKKK, Dhc-SKKKK and FSL-1 (Pam2CGDPKHPKSF) Anticancer vaccine composition, characterized in that at least one selected from the group consisting of.
According to claim 7,
The liposome (liposome) is an anti-cancer vaccine composition, characterized in that composed of lipids.
According to claim 9,
The lipid is DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DDA (Dimethyldioctadecylammonium), DC-chol (3β-[N-( N',N'-Dimethylaminoethane)-carbamoyl]cholesterol), DOPG (1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)]), DPPC (1,2-dipalmitoyl-sn An anti-cancer vaccine composition, characterized in that at least one selected from the group consisting of -glycero-3phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and cholesterol.
According to claim 2,
The adjuvant is an anti-cancer vaccine composition, characterized in that composed of a liposome having a positive charge in which a lipopeptide having a positive charge is inserted into a lipid bilayer and an immunoactive substance having a negative charge.
According to claim 2,
The immunoactive substance is poly (I: C), QS21, MPLA (Monophosphoryl Lipid A), CpG and flagellin (Flagellin) characterized in that at least one selected from the group consisting of anti-cancer vaccine composition.
According to claim 12,
The poly (I: C) is an anti-cancer vaccine composition, characterized in that the length is 50 to 5,000 bp.
According to claim 6,
The mixture includes lipopeptide and poly(I:C) in a composition ratio of 1.25:1 to 2:1, wherein the lipopeptide is 0.01% to 0.1% by weight; and poly(I:C) in a weight ratio of 0.01% to 0.08% by weight.
According to claim 1 or 2,
The anti-cancer vaccine composition is an anti-cancer vaccine composition characterized in that it induces a humoral immune response and a cellular immune response.
According to claim 1 or 2,
The anti-cancer vaccine composition is an anti-cancer vaccine composition characterized in that it enhances the Th1 immune response.
According to claim 15,
The humoral immune response is an anti-cancer vaccine composition, characterized in that by increasing T follicular helper cell (Tfh) to secrete cytokines.
According to claim 15,
The cellular immune response is an anti-cancer vaccine composition, characterized in that the secretion of IFN-γ, TNF-α and granzyme B is enhanced.
According to claim 1 or 2,
The composition is an anticancer vaccine composition, characterized in that the formulation is an aqueous solution formulation.
A pharmaceutical composition for preventing or treating cancer, comprising the anti-cancer vaccine composition of claim 1 or 2 as an active ingredient.
According to claim 19,
The cancers include ovarian cancer, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, intracranial race, ependymoma, brainstem tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus cancer, nasopharyngeal cancer, salivary gland cancer, Hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinum tumor, esophageal cancer, breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer , Bladder cancer, kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, uterine sarcoma, vaginal cancer, female external genital cancer, and skin cancer. composition.
According to claim 19,
The anti-cancer vaccine composition is characterized in that it is used in combination with one or more selected from the group consisting of chemotherapy, targeted therapy, immune checkpoint inhibitor, and radiation therapy. A pharmaceutical composition for prevention or treatment.

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